1. Field of the Invention
This invention relates to TOM1 gene and TOM3 gene, which are host-encoded protein factors derived from Arabidopsis thaliana indispensable for the multiplication of a plant virus and relates to a transgenic plant wherein its virus resistance is enhanced.
2. Description of Related Art
Virus replication is suggested to take place through specific interaction among viral replication proteins, viral RNAs and host-encoded proteins. However, any host-encoded plant genes indispensable for the multiplication of viruses have not been identified.
Heretofore, a technique utilizing hypersensitive reaction have been adopted to render virus resistance to a plant. Concerning the technique, following problems should be solved prior to practical use.
(1) The virus resistance can not be rendered to a plant of other species.
(2) The virus resistance is only available for limited range of viruses.
(3) The frequent emergence of a mutated virus strain, which can conquer virus resistance of the host plant is hazardous biologically.
On the other hand, expression of a part of virus genome in a plant body to render virus resistance have came to a stage of practical use, following problems should be solved.
(1) The virus resistance is only available for limited range of viruses.
(2) The possible emergence of a novel virus strain by gene recombination is hazardous biologically.
The inventors have cloned Arabidopsis thaliana TOM1 and TOM3 genes, which encode proteins indispensable for the multiplication of a plant virus. The virus resistance might be rendered to a plant by expression regulation or alteration of TOM1 or TOM3 gene.
In anti-viral strategies utilized so far, many methods have not been used universally, because engineered virus resistance was specific to the virus species. The TOM1 gene of Arabidopsis thaliana was selected as a target gene, because the gene is a host plant-encoded gene indispensable for multiplication of a virus in a plant body. Therefore, it is assumed that, a gene with high homology would exist in other plant species and other alpha-like viruses of plants or animals would multiplicate using certain product of host-encoded gene similar to TOM1. From the assumption, production of an organism with virus resistance by manipulation of TOM1 gene or its homolog may be effective to resist against various kinds of alpha-like viruses. Moreover, emergence of a mutated virus strain which can conquer virus resistance of the host plant is not likely to occur, for this method inhibits essential interaction between the host plant and the virus.
In this invention, a mutant strain of Arabidopsis thaliana, with decreased accumulation of TMV-Cg CP by virus infection, was produced. The TOM1 gene, indispensable for the efficient multiplication of TMV, was obtained from the wild-type strain by positional cloning. Moreover, a nucleotide sequence of TOM1 gene and an amino acid sequence of TOM1 protein were determined. A double mutant strain of Arabidopsis thaliana was obtained by further mutagenesis of the tom1 mutant. In the double mutant strain, the activity to multiplicate tabacco mosaic virus remained in the tom1 mutant strain is completely abolished. TOM3 gene, which is indispensable for the multiplication of TMV in tom1 mutant, was obtained, and a nucleotide sequence of TOM3 gene and an amino acid sequence of TOM3 protein were determined. The anti-viral strategy of this invention is free from hazard of emergence of a novel virus strain by gene recombination, for gene manipulation of this method is limited to host plant-encoded gene. Therefore, the anti-viral strategy of this invention is expected to be effective and free from biological hazard, and this strategy would be of great application on agricultural or medical field.
The TOM1 gene of this invention is identified by a nucleotide sequence referred to sequence number 2 in a sequence list. The TOM1 protein, a polypeptide encoded by the TOM1 gene, is identified by an amino acid sequence referred to sequence number 1 in a sequence list. Moreover, the TOM3 gene of this invention is identified by a nucleotide sequence referred to sequence number 4 in a sequence list. The TOM3 protein, a polypeptide encoded by the TOM3 gene, is identified by an amino acid sequence referred to sequence number 3 in a sequence list. As described above, the TOM1 gene and the TOM3 gene are originated from Arabidopsis thaliana and the TOM1 protein and the TOM3 protein are host-encoded plant factors indispensable for multiplication of tabacco mosaic virus. Thus, virus resistance can be rendered to a plant, by expression regulation or alteration of the genes of this invention and the genes of this invention can be used for such purpose. For example, anti-sense RNA of TOM1, TOM3 or both genes would be available, and expression regulation of TOM1, TOM3 or both genes by using the technique of co-suppression would be also available. Moreover, virus resistance can be rendered to a plant, by incorporation of mutated tom1, tom3 or both genes encoding proteins capable of interacting with a virus but not capable of supporting multiplication of a virus. Such mutation can cause dominant-negative effect on the function of TOM1, TOM3 or both genes.
In general, one amino acid is encoded by plural base sequences of DNA. Therefore, plural genes, other than native gene of this invention, might encode amino acid sequence identical to TOM1 or TOM3 protein. The genes of this invention are not to be limited to only native genes and intended to include many other base sequences encoding TOM1 or TOM3 protein.
The TOM1 protein of this invention includes a polypeptide having an amino acid sequence at least 50% of sequence homology with the amino acid sequence referred to sequence number 1 in a sequence list, as far as having the activity to support the multiplication of alpha-like virus. In preferred form, the precursor polypeptide of this invention have more than 70% of sequence homology with the amino acid sequence referred to sequence number 1 in a sequence list. In more preferred form, the precursor polypeptide of this invention have more than 90% of sequence homology with the amino acid sequence referred to sequence number 1 in a sequence list.
In the same manner, The TOM3 protein of this invention includes a polypeptide having an amino acid sequence at least 50% of sequence homology with the amino acid sequence referred to sequence number 3 in a sequence list, as far as having the activity to multiplicate alpha-like virus. In preferred form, the precursor polypeptide of this invention have more than 70% of sequence homology with the amino acid sequence referred to sequence number 3 in a sequence list. In more preferred form, the precursor polypeptide of this invention have more than 90% of sequence homology with the amino acid sequence referred to sequence number 3 in a sequence list.
Using the recombinant DNA technique, a DNA can be mutated artificially at a certain position without alteration or with improvement of basic characteristic of a protein encoded by the nucleotide sequence of the DNA. Concerning genes having native nucleotide sequences provided by this invention, and other genes having base sequences encoding TOM1 or TOM3 protein, these sequences can be altered artificially by insertion, deletion or substitution of the sequences, so far as retaining characteristic equivalent or improved compared to the native proteins. It is to be understood that mutated genes described above are in the range of this invention.
Therefore, the gene of this invention includes a gene that encodes the TOM1 protein described above consisting of a base sequence that hybridizes with the nucleotide sequence referred to sequence number 2 in a sequence list under a contingent condition.
In the same manner, the gene of this invention includes a gene that encodes the TOM3 protein described above consisting of a nucleotide sequence that hybridizes with the base sequence referred to sequence number 4 in a sequence list under a contingent condition.
Moreover, a transgenic plant wherein the gene of this invention is incorporated is also in the range of this invention. For the examples of plants preferable for incorporation of the genes of this invention, monocotyledonous plants such as rice, barley, wheat or corn and dicotyledonous plants such as eggplant, tomato and potato can be mentioned. For the examples of methods preferred to be adopted for incorporation of genes of this invention, agrobacterium method, protoplast-PEG method, protoplast-electroporation method, particle gun method and micro-injection method can be mentioned. This invention will be described in more details by following embodiment, above description and below embodiment are not intended to limit the range of this invention and intended to include ordinal alteration in the art.